Specific Aim 3 (SA3) describes molecular genetic characterization of likely synapsis proteins c(3)G and dmSmc3p. Both of these proteins have been cloned and are ready for genetic and immunological experiments that will determine function and protein localization. In SA4, Dr. Hawley will use a clever genetic screen to find new genes required for interstitial synapsis function. The genetic background suppresses telomere and centromeric heterochromatin-dependent synapsis, making all X-chromosome crossover dependent on interstitial sites. Dr. Hawley's expertise with meiotic screens means that this otherwise difficult screen will produce useful results. SA1 & 2 focus on the experiments that will follow synapsis and crossover by direct observation of loci on oocyte chromosomes marked with molecular probes. In SA2, in situ hybridization with P1 clones will serve as markers of X-chromosome sites within specific interstitial segments. Homologous site pairing will be explored in various mutant backgrounds that Dr. Hawley's model predicts should disrupt pairing efficiency. These experiments will address several significant questions including the validity of the "synapsis domain" paradigm proposed by the Hawley model and if episomal pairing sites antagonize pairing efficiency of the homologous chromosomal sites. Similar experiments (SA1) will examine oocyte chromosomes for evidence of meiotic bouquet formation, the aggregation of telomeres/centromeres within special nuclear domains. Such bouquets may play a role in chromosome alignment that precedes synapsis. In SA1, Dr. Hawley proposes to use newly developed (and developing) imaging methods to view live oocyte chromosomes during meiosis. These experiments will be conducted in collaboration with Dr. J. Sedat. Chromosomes will be marked in vivo with lacI:GFP protein that will bind to lacO sites inserted into fly chromosomes by germline transformation. Real-time data of concordance/discordance of specific sites (telomeres, centromeres, interstitial pairing sites) will reveal dynamic features of chromosome arrangements during specific meiotic prophase stages. In turn, those data will address many outstanding questions about how synapsis and recombination is accomplished.